Reinforced absorbable multilayered fabric for use in tissue repair and regeneration

ABSTRACT

The present invention is directed to a method of using a multilayered fabric comprising a first absorbable nonwoven fabric and a second absorbable woven or knitted fabric in tissue repair and regeneration.

This application claims priority from U.S. Provisional Application Ser.No. 60/620,624, filed on 20 Oct. 2004.

FIELD OF THE INVENTION

The present invention relates to a method for using a reinforcedabsorbable multilayered fabric in tissue repair and regeneration.

BACKGROUND OF THE INVENTION

It is generally know to use multilayered fabrics in connection withmedical procedures. For example, multilayered fabrics are used as allpurpose pads, wound dressings, surgical meshes, including hernia repairmeshes, adhesion prevention meshes and tissue reinforcement meshes,defect closure devices, and hemostats.

For example, U.S. Pat. No. 5,593,441 to Lichtenstein et al describes acomposite prosthesis preferably having a sheet of polypropylene meshthat allows tissue in-growth, such as Marlex® mesh. This referencediscloses that other surgical materials that are suitable for tissuereinforcement and defect closure may be utilized, including absorbablemeshes such as a polyglactin 910 (Vicryl®) mesh. The compositeprosthesis of Lichtenstein et al also has an adhesion barrier,preferably a sheet of silicone elastomer. This reference generallysuggests that that an oxidized regenerated cellulose such as Interceed®(TC7) absorbable adhesion barrier (commercially available from Ethicon,Inc., in Somerville, N.J.) may be used as the adhesion barrier toproduce a composite prosthesis having short term effectiveness. Thecomposite prosthesis of Lichtenstein et al is described for use inreinforcing and repairing a weakened muscular wall while limiting theincidence of postoperative adhesions.

U.S. Pat. No. 5,686,090 to Schilder et al describes the use of a fleecein combination with a nonabsorbable or absorbable film to preventmis-growths to adjacent tissue and to reduce adhesions. Schilder et algenerally discloses that polypropylene, polyester, polyglactin,polydioxanone or poliglecaprone 25 may be used as the fleece material orthe film material. The term “fleece” as used in this reference isdescribed by its porosity, which is described as being in the rangebetween 100 and 1000 l/(m²s) gas flow, measured with an inlet pressureof 200 Pa, a test surface of 50 cm² and a test thickness of 1 mm. Thecomposite of Schilder et al is generally described as being amultilayered implant.

Additionally, multilayered fabrics are useful for tissue engineering andorthopedic applications. The recent emergence of tissue engineeringoffers numerous approaches to repair and regenerate damaged/diseasedtissue. Tissue engineering strategies have explored the use ofbiomaterials in combination with cells and/or growth factors to developbiological substitutes that ultimately can restore or improve tissuefunction. The use of colonizable and remodelable scaffolding materialshas been studied extensively as tissue templates, conduits, barriers,and reservoirs. In particular, synthetic and natural materials in theform of foams, sponges, gels, hydrogels, textiles, and nonwovens havebeen used in vitro and in vivo to reconstruct/regenerate biologicaltissue, as well as deliver chemotactic agents for inducing tissuegrowth. The different forms of scaffolds may be laminated to form amultilayered tissue engineering scaffold.

However, the prior art fails to describe or suggest a reinforcedabsorbable multilayered fabric having a first absorbable nonwoven fabricreinforced by a second absorbable woven or knitted fabric.

As used herein, the term “nonwoven fabric” includes, but is not limitedto, bonded fabrics, formed fabrics, or engineered fabrics, that aremanufactured by processes other than spinning, weaving or knitting. Morespecifically, the term “nonwoven fabric” refers to a porous,textile-like material, usually in flat sheet form, composed primarily orentirely of staple fibers assembled in a web, sheet or batt. Thestructure of the nonwoven fabric is based on the arrangement of, forexample, staple fibers that are typically arranged more or lessrandomly. The tensile, stress-strain and tactile properties of thenonwoven fabric ordinarily stem from fiber to fiber friction created byentanglement and reinforcement of, for example, staple fibers, and/orfrom adhesive, chemical or physical bonding. Notwithstanding, the rawmaterials used to manufacture the nonwoven fabric may be yarns, scrims,netting, or filaments made by processes that include spinning, weavingor knitting.

SUMMARY OF THE INVENTION

The present invention is directed to a method of using a reinforcedabsorbable multilayered fabric comprising a first absorbable nonwovenfabric reinforced by a second absorbable woven or knitted fabric fortissue repair and regeneration. The method comprises first preparing arepair site for implantation, and then disposing the reinforcedabsorbable multilayered fabric at the site. The first absorbablenonwoven fabric comprises fibers comprising aliphatic polyesterpolymers, copolymers, or blends thereof; while the second absorbablewoven or knitted fabric comprises oxidized regenerated cellulose fibers.

DETAILED DESCRIPTION OF THE INVENTION

The reinforced absorbable multilayered fabric generally comprises anonwoven fabric and a reinforcement fabric. The reinforcement fabricprovides a backing to which the nonwoven fabric may be attached, eitherdirectly or indirectly.

The nonwoven fabric functions as the first absorbable nonwoven fabric ofthe reinforced absorbable multilayered fabric described herein. Thefirst absorbable nonwoven fabric is comprised of fibers comprisingaliphatic polyester polymers, copolymers, or blends thereof. Thealiphatic polyesters are typically synthesized in a ring openingpolymerization of monomers include but are not limited to lactic acid,lactide (including L-, D-, meso and D, L mixtures), glycolic acid,glycolide, ε-caprolactone, p-dioxanone (1,4-dioxan-2-one), andtrimethylene carbonate (1,3-dioxan-2-one).

Preferably, the first absorbable nonwoven fabric comprises a copolymerof glycolide and lactide, in an amount ranging from 70 to 95% by molarbasis glycolide and the remainder lactide.

In an alternative embodiment, the first absorbable nonwoven fabriccomprises fibers comprised of aliphatic polyester polymers, copolymers,or blends in combination with oxidized polysaccharide fibers.

Preferably, the nonwoven fabric is made by processes other thanspinning, weaving or knitting. For example, the nonwoven fabric may beprepared from yarn, scrims, netting or filaments that have been made byprocesses that include spinning, weaving or knitting. The yarn, scrims,netting and/or filaments are crimped to enhance entanglement with eachother and attachment to the second absorbable woven or knitted fabric.Such yarn, scrims, netting and/or filaments may be cut into staple thatis long enough to entangle. The staple may be between about 0.1 and 3.0inches long, preferably between about 0.75 and 2.5 inches, and mostpreferably between about 1.5 and 2.0 inches. The staple may be carded tocreate a nonwoven batt, which may be then needlepunched or calendaredinto the first absorbable nonwoven fabric. Additionally, the staple maybe kinked or piled.

Other methods known for the production of nonwoven fabrics may beutilized and include such processes as air laying, wet forming andstitch bonding. Such procedures are generally discussed in theEncyclopedia of Polymer Science and Engineering, Vol. 10, pp. 204-253(1987) and Introduction to Nonwovens by Albin Turbank (Tappi Press,Atlanta Ga. 1999), both incorporated herein in their entirety byreference.

The thickness of the nonwoven fabric may range from about 0.25 to 2 mm.The basis weight of the nonwoven fabric ranges from about 0.01 to 0.2g/in²; preferably from about 0.03 to 0.1 g/in²; and most preferably fromabout 0.04 to 0.08 g/in². The weight percent of first absorbablenonwoven fabric may range from about 10 to 80 percent, based upon thetotal weight of the reinforced absorbable multilayered fabric.

The second absorbable woven or knitted fabric functions as thereinforcement fabric and comprises oxidized polysaccharides, inparticular oxidized cellulose and the neutralized derivatives thereof.For example, the cellulose may be carboxylic-oxidized oraldehyde-oxidized cellulose. More preferably, oxidized regeneratedpolysaccharides including, but without limitation, oxidized regeneratedcellulose may be used to prepare the second absorbable woven or knittedfabric. Regenerated cellulose is preferred due to its higher degree ofuniformity versus cellulose that has not been regenerated. Regeneratedcellulose and a detailed description of how to make oxidized regeneratedcellulose is set forth in U.S. Pat. Nos. 3,364,200, 5,180,398, and4,626,253, the contents each of which is hereby incorporated byreference as if set forth in its entirety.

Examples of fabrics that may be utilized as the reinforcement fabricinclude, but are not limited to, Interceed® absorbable adhesion barrier,Surgicel® absorbable hemostat; Surgicel Nu-Knit® absorbable hemostat;and Surgicel® Fibrillar absorbable hemostat; each available from Johnson& Johnson Wound Management Worldwide or Gynecare Worldwide, each adivision of Ethicon, Inc., Somerville, N.J.

The reinforcement fabric utilized in the present invention may be wovenor knitted, provided that the fabric possesses the physical propertiesnecessary for use in contemplated applications. Such fabrics, forexample, are described in U.S. Pat. Nos. 4,626,253, 5,002,551 and5,007,916, the contents of which are hereby incorporated by referenceherein as if set forth in its entirety. In preferred embodiments, thereinforcement fabric is a warp knitted tricot fabric constructed ofbright rayon yarn that is subsequently oxidized to include carboxyl oraldehyde moieties in amounts effective to provide the fabrics withbiodegradability.

In an alternative embodiment, the second absorbable woven or knittedfabric comprises oxidized polysaccharide fibers in combination withfibers comprised of aliphatic polyester polymers, copolymers, or blendsthereof.

The second absorbable woven or knitted fabric preferably comprisesoxidized regenerated cellulose and may have a basis weight ranging fromabout 0.001 to 0.2 g/in², preferably in the range of about 0.01 to 0.1g/in², and most preferably in the range of about 0.04 to 0.07 g/in².

The first absorbable nonwoven fabric is attached to the secondabsorbable woven or knitted fabric, either directly or indirectly. Forexample, the nonwoven fabric may be incorporated into the secondabsorbable woven or knitted fabric via needlepunching, calendaring,embossing or hydroentanglement, or chemical or thermal bonding. Thestaple of the first absorbable nonwoven fabric may be entangled witheach other and imbedded in the second absorbable woven or knittedfabric. More particularly, for methods other than chemical or thermalbonding, the first absorbable nonwoven fabric may be attached to thesecond absorbable woven or knitted fabric such that at least about 1% ofthe staple of the first absorbable nonwoven fabric are exposed on theother side of the second absorbable woven or knitted fabric, preferablyabout 10-20% and preferably no greater than about 50%. This ensures thatthe first absorbable nonwoven fabric and the second absorbable woven orknitted fabric remain joined and do not delaminate under normal handlingconditions. The reinforced absorbable multilayered fabric is uniformsuch that substantially none of the second absorbable woven or knittedfabric is visibly devoid of coverage by the first absorbable nonwovenfabric.

One method of making the multilayered fabric described herein is by thefollowing process. Absorbable polymer fibers, having a denier per fiberof about 1 to 4, may be consolidated to about 80 to 120 deniermultifilament yarn and then to about 800 to 1200 denier yarns, thermallycrimped and then cut to a staple having a length between about 0.75 and2.5 inch. The staple may be fed into a multiroller dry lay cardingmachine one or more times and carded into a uniform nonwoven batt, whilehumidity is controlled between about 40-60% at a room temperature of 60to 75° C. The batt may be further processed via needlepunching or anyother means such as calendaring. Thereafter, the first absorbablenonwoven fabric may be attached to the second absorbable woven orknitted fabric by various techniques such as needlepunching. Thereinforced absorbable multilayered fabric may then be scoured by washingin an appropriate solvent and dried under mild conditions forapproximately 30 minutes.

It is desirable to control process parameters such as staple length,opening of the staple, staple feed rate, and relative humidity. Forexample, the consolidated yarns may have from about 5 to 50 crimps perinch and preferably from about 10 to 30 crimps per inch. Efficientcutting of the crimped yarns is desirable, as any long and incompletelycut staple tends to stick on the carding machine and cause pilling. Apreferred range of the staple length is from about 0.75 to 2.5 inches,and preferably from about 1.5 to 2.0 inches.

To optimize uniformity and minimize the build-up of static electricity,the relative humidity may be controlled during batt processing,preferably during carding to form the uniform nonwoven batt. Preferably,the nonwoven batt is processed using a dry lay carding process at arelative humidity of at least about 40% at a room temperature of about60 to 75° C. More preferably, the nonwoven batt is processed at arelative humidity of from about 50% to 60%.

The multilayered fabric is scoured using solvents suitable to dissolveany spin finish. Solvents include, but are not limited to, isopropylalcohol, hexane, ethyl acetate, and methylene chloride. The multilayeredfabric is then dried under conditions to provide sufficient drying whileminimizing shrinkage.

The reinforced absorbable multilayered fabric may have an averagethickness of between about 0.75 and 3.0 mm, preferably between about1.00 and 2.5 mm, and most preferably between about 1.2 and 2.0 mm. Thebasis weight of the reinforced absorbable multilayered fabric is betweenabout 0.05 and 0.25 g/in², preferably between about 0.08 and 0.2 g/in²,and most preferably between about 0.1 and 0.18 g/in². The reinforcedabsorbable multilayered fabric is uniform such that there is no morethan about 10% variation (relative standard deviation of the mean) inthe basis weight or thickness across each square inch of the.

Additionally, the reinforced absorbable multilayered fabric may containbioactive agents to aid in the repair or regeneration of tissue.Examples of bioactive agents include cell attachment mediators, such aspeptide-containing variations of the “RGD” integrin binding sequenceknown to affect cellular attachment, biologically active ligands, andsubstances that enhance or exclude particular varieties of cellular ortissue ingrowth. Examples of such substances include integrin bindingsequence, ligands, bone morphogenic proteins, epidermal growth factor,IGF-I, IGF-II, TGF-β I-III, growth differentiation factor, parathyroidhormone, vascular endothelial growth factor, hyaluronic acid,glycoprotein, lipoprotein, bFGF, TGF-beta superfamily factors, BMP-2,BMP-4, BMP-6, BMP-12, sonic hedgehog, GDF5, GDF6, GDF8, PDGF,recombinant human growth and differentiation factor 5 (rhGDF-5), smallmolecules that affect the upregulation of specific growth factors,tenascin-C, fibronectin, thromboelastin, thrombin-derived peptides,heparin-binding domains, and the like.

Bioactive agents may also include biologically derived substanceselected from the group consisting of demineralized bone matrix (DBM),platelet rich plasma, bone marrow aspirate and bone fragments, all ofwhich may be from autogenic, allogenic, or xenogenic sources.

In other embodiments, the reinforced absorbable multilayered fabric maycontain an inorganic filler. The inorganic filler may be selected fromalpha-tricalcium phosphate, beta-tricalcium phosphate, calciumcarbonate, barium carbonate, calcium sulfate, barium sulfate,hydroxyapatite, and mixtures thereof. In certain embodiments theinorganic filler comprises a polymorph of calcium phosphate such as aphosphate glass.

In yet another embodiment, the reinforced absorbable multilayered fabriccan be seeded or cultured with appropriate cell types prior toimplantation for the targeted tissue. Cells which can be seeded orcultured on the matrices of the current invention include, but are notlimited to, bone marrow cells, stromal cells, stem cells, embryonic stemcells, chondrocytes, osteoblasts, osteocytes, osteoclasts, fibroblasts,pluripotent cells, chondrocyte progenitors, endothelial cells,macrophages, leukocytes, 5 adipocytes, monocytes, plasma cells, mastcells, umbilical cord cells, postpartum derived cells, mesenchymal stemcells, epithelial cells, myoblasts, and precursor cells derived fromadipose tissue.

The cells can be seeded on the multilayered fabric for a short period oftime, e.g. less than one day, just prior to implantation, or culturedfor longer a period, e.g. greater than one day, to allow for cellproliferation and matrix synthesis within the seeded fabric prior toimplantation.

The reinforced absorbable multilayered fabric can be used as a scaffoldin tissue repair and regeneration applications. Examples of tissuerepair and regeneration include rotator cuff repair, repair of damagedknee cartilage, and regeneration of skin, muscle, or bone lost due todisease or injury.

The method for using the reinforced absorbable multilayered fabric isto: first prepare a repair site for implantation, and then dispose thefabric at the site of repair. The fabric may be attached to the site ofrepair with sutures, tacks, or any of a number of biocompatible glues.

EXAMPLE 1 Nonwoven PGL Fabric with ORC Fabric

Poly (glycolide-co-lactide) (PGL, 90/10 mol/mol) was melt-spun intofiber. A multi-filament yarn was consolidated, crimped and cut intostaple having a length of 1.75 inches. The staple was carded to create anonwoven batt and then compacted to a thickness of about 1.25 mm and adensity of about 98.1 mg/cc. The nonwoven fabric was then needlepunchedinto a knitted carboxylic-oxidized regenerated cellulose (ORC) fabric,available from Ethicon, Inc., under the tradename Interceed®, to securethe nonwoven fabric to the ORC fabric. The final multilayered fabriccomprised about 60 weight percent of the nonwoven fabric.

EXAMPLE 2 Nonwoven PGL Fabric with ORC Fabric

Poly (glycolide-co-lactide) (PGL, 90/10 mol/mol) was melt-spun intofiber. A multi-filament yarn was consolidated, crimped and cut intostaple having a length of 1.75 inches. The staple was carded to create anonwoven batt and then compacted to a thickness of about 1.22 mm and adensity of about 103.4 mg/cc. The nonwoven fabric was then needlepunchedinto a knitted carboxylic-oxidized regenerated cellulose fabric (ORC),available from Ethicon, Inc., under the tradename Surgicel NuKnit®, tosecure the nonwoven fabric to the ORC fabric. The final multilayeredfabric comprised about 25 weight percent of the nonwoven fabric.

EXAMPLE 3 Nonwoven PGL Fabric with ORC Fabric

Poly (glycolide-co-lactide) (PGL, 90/10 mol/mol) was melt-spun intofiber. A multi-filament yarn was consolidated, crimped and cut intostaple having a length of 1.75 inches. The staple was carded to create anonwoven batt and then compacted a felt having a thickness of about 1.1mm and a density of about 102.8 mg/cc. The nonwoven fabric was thenneedlepunched into a knitted carboxylic-oxidized regenerated cellulosefabric (ORC), available from Ethicon, Inc., under the tradenameSurgicel®, to secure the nonwoven fabric to the ORC fabric. The finalmultilayered fabric comprised about 60 weight percent of the nonwovenfabric.

EXAMPLE 4 Nonwoven PGL Fabric with ORC Fabric

Poly (glycolide-co-lactide) (PGL, 90/10 mol/mol) was melt-spun intofiber. A 80 denier multifilament yarn was consolidated into a 800 denierconsolidated yarn. The consolidated yarn was crimped at approximately110 degree C. The crimped yarn was cut into staple having a length ofabout 1.25″ in length. 20 g of the crimped staple was accurately weighedand laid out uniformly on the feed conveyor belt of a multi-rollercarding machine. The environmental conditions (temp: 70 deg F./55% RH)were controlled. The staple was then carded to create a nonwoven batt.The batt was removed from the pick-up roller and cut into 4 equal parts.These were re-fed into the carder perpendicular to the collectiondirection. After this second pass the batt was weighed (19.8 g: 99%fabric yield) and then compacted into a felt. The compact felt wasprecisely laid onto an ORC fabric and firmly attached via 2 passes inthe needlepunching equipment. The multilayered fabric was trimmed andscoured in 3 discrete isopropyl alcohol baths to remove spin finish andany machine oils. The scoured multilayered fabric was dried in an ovenat 70 degree C. for 30 minutes, cooled and weighed.

The “thickness” of the multilayered fabric was measured as describedherein. The measurement tools were:

-   (1) Mitutoyo Absolute gauge Model number ID-C125EB [Code    number—543-452B]. The 1″ diameter foot was used on the gauge.-   (2) A magnetic holder was used to lock in place and set the caliper    up to the die platen.-   (3) Two metal plates ˜2.75″×2″×0.60″, weighing between 40.8 g to    41.5 g [combined total of ˜82.18 g].    The multilayered fabric was placed on a platen surface that is a    smooth and machined surface. The two metal plates were placed on top    of each other on the multilayered fabric and gently pressed at their    corners to make sure the multilayered fabric is flat. The gauge foot    was placed onto the top of the metal plates and was then re-lifted    and re-placed, at which time a reading was made.    12—1″×1″ pieces were die-cut from the scoured multilayered fabric    and accurately weighed. The thickness of each 1″×1″ piece was    measured 4-5 times in different areas of the metal plate in order to    obtain a reliable average. The weight and thickness of each piece is    shown in Table 1. The values indicate that the coverage of both    layers is similar in all directions.

TABLE 1 Sheet #1 Sheet #2 Sample # Weight (g) Thickness (mm) Weight (g)Thickness (mm)  1 .132 1.53 .13 1.58  2 .132 1.58 .124 1.57  3 .131 1.59.13 1.62  4 .129 1.55 .134 1.64  5 .126 1.58 .126 1.56  6 .125 1.5 .1311.59  7 .129 1.56 .136 1.7  8 .127 1.52 .131 1.62  9 .132 1.55 .131 1.5710 .123 1.58 .136 1.58 11 .128 1.58 .135 1.65 12 .13 1.51 .133 1.55Average 0.1287 1.5525 0.1314 1.6025 Std. Dev 0.0029 0.031 0.0037 0.044CV (%) 2.304 2.002 2.837 2.767

EXAMPLE 5 Rotator Cuff Repair Using Reinforced Absorbable MultilayeredFabric

In the case of a rotator cuff problem, the surgeon first looks at theextent of an injury using an arthroscope. Then, under generalanesthesia, the patient undergoes open surgery to repair the tear.

After the anesthetic has been administered and the shoulder has beenprepared, a cosmetic incision is made over the top front corner of theshoulder. This incision allows access to the seam between the front andmiddle parts of the deltoid muscle. Splitting this seam allows access tothe rotator cuff without detaching or damaging the important deltoidmuscle, which is responsible for a significant portion of the shoulder'spower. All scar tissue is removed from the space beneath the deltoid andthe acromion (part of the shoulder blade to which the deltoid attaches).Thickened bursa and the rough edges of the rotator cuff and humerus(upper arm bone) are also smoothed to make sure that they pass smoothlybeneath the acromion and deltoid.

The edges of the cuff tendons are identified and the quality andquantity of the cuff tissue is determined. The goal of the repair is toreattach good quality tendon to the location on the arm bone from whichit was torn. A groove or trough is fashioned in the normal attachmentsite for the cuff. To support the tendon and aid in healing, the surgeonsutures a patch of reinforced absorbable multilayered fabric into placeover it. Sutures (lengths of surgical thread) draw the edge of thetendon securely into the groove to which it is to heal.

The surgeon then completes the surgery by closing the deltoid muscle andthe skin incision. Over time, the body creates new tissue in the areathat matches surrounding tissue. The body also absorbs the implantedpatch in two to four months.

EXAMPLE 6 Knee Cartilage Repair Using Reinforced Absorbable MultilayeredFabric

First, the surgeon examines the knee through an arthroscope—a smalldevice that allows the doctor to see into your knee joint. If a lesionis detected, a surgical procedure is performed.

After the anesthetic has been administered and the knee has beenprepared, a cosmetic incision is made through the skin over the topfront corner of the patella. First, the damaged cartilage is removed.The reinforced absorbable multilayered fabric is then implanted into thelesion. The fabric may be attached to the lesion site with sutures,tacks, or any of a number of biocompatible glues.

The surgeon then completes the surgery by closing the skin incision.Cartilage cells migrate into and multiply in the implanted fabric, andthe cell/fabric implant intergrates with surrounding cartilage. Withtime, the cells will mature and fill-in the lesion with hyalinecartilage.

EXAMPLE 7 Tissue Repair Using Reinforced Absorbable Multilayered Fabric

Goat cartilage was harvested from femoral condyles of 1 to 1½ year oldgoat knees. A first set of 5-millimeter diameter discs were made fromthe cartilage using a punch. The cartilage discs were washed inphosphate buffered saline (PBS) containing antibiotic and antimycotics,then rinsed in PBS. Separately, a second set of 5-millimeter diameterdiscs were made from ORC/Polyglactin multilayered fabric formed asdescribed in Example 1 and sterilized by Ethylene Oxide (EtO).

The fabric discs were placed in between two cartilage discs and bondedtogether with bovine fibrin glue. Constructs were implantedsubcutaneously in SCID mice. Six weeks following implantation, sampleswere collected and processed for histology. Histological slides wereassessed for repair tissue in the fabric discs by Hematoxylin/Eosin andSafranin O staining.

Results showed that cells had migrated into the scaffolds and formed newrepair tissue. At this time point the repair tissue was fibrous innature.

EXAMPLE 8 Tissue Repair Using Reinforced Absorbable Multilayered FabricHaving a Bioactive Agent

Constructs for SCID implantation were prepared similar to that describedfor Example 9 with the exception that the ORC/Polyglactin compositeswere loaded with 2.5 micrograms of recombinant human growth anddifferentiation factor 5 (rhGDF-5) and lyophilized. Constructs wereimplanted in SCID mice and processed similar to Example 7. Histologicalslides were assessed for repair tissue in scaffolds by Hematoxylin/Eosinand Safranin O staining.

Results showed that cells had migrated into the scaffolds and formed newrepair tissue. In the presence of the growth factor new cartilage likerepair tissue was formed within the scaffolds. This repair tissuestained positive for Safranin O a stain specific for sulfatedproteoglycans.

While the examples demonstrate certain embodiments of the invention,they are not to be interpreted as limiting the scope of the invention,but rather as contributing to a complete description of the invention.All reinforcement fabrics described in the examples below are thenonsterile materials of the corresponding commercial products referredby their tradenames.

1. A method of tissue repair and regeneration, said method comprising:preparing a repair site for implantation, providing a reinforcedabsorbable multilayered fabric, said fabric comprising: a firstabsorbable nonwoven fabric comprising staple having a length from about1.5 to 2 inches, and a second absorbable woven or knitted fabric, anddisposing said fabric at said site.
 2. The method of claim 1, where saidreinforced absorbable multilayered fabric is attached to said site ofrepair with suture, tack, or biocompatible glue.
 3. The method of claim1, where said first absorbable nonwoven fabric of said multilayeredfabric comprises fibers comprised of aliphatic polyester polymers orcopolymers of one or more monomers selected from the group consisting oflactic acid, lactide (including L-, D-, meso and D, L mixtures),glycolic acid, glycolide, .epsilon.-caprolactone, p-dioxanone, andtrimethylene carbonate.
 4. The method of claim 3, where said firstabsorbable nonwoven fabric comprises glycolide/lactide copolymer.
 5. Themethod of claim 3, where said second absorbable woven or knitted fabricof said multilayered fabric comprises oxidized polysaccharides.
 6. Themethod of claim 5, where said second absorbable woven or knitted fabriccomprises oxidized cellulose.
 7. The method of claim 5, where saidsecond absorbable woven or knitted fabric comprises oxidized regeneratedcellulose.
 8. The method of claim 1, where said first absorbablenonwoven fabric comprises glycolide/lactide copolymer, and said secondabsorbable woven or knitted fabric comprises oxidized regeneratedcellulose.
 9. The method of claim 1, where said staple is crimped. 10.The method of claim 1, where said staple is derived from fiber of about1 to 4 denier per filament.
 11. The method of claim 10, where said firstabsorbable nonwoven fabric has a basis weight of about 0.01 to 0.2g/in.sup.2; and said second absorbable woven or knitted fabric has abasis weight of about 0.001 to 0.2 g/in.sup.2.
 12. The method of claim1, where said reinforced absorbable multilayered fabric furthercomprises a bioactive agent selected from the group consisting ofintegrin binding sequence, ligands, bone morphogenic proteins, epidermalgrowth factor, IGF-I, IGF-II, TGF-.beta. I-Ill, growth differentiationfactor, parathyroid hormone, vascular endothelial growth factor,hyaluronic acid, glycoprotein, lipoprotein, bFGF, TGF-beta superfamilyfactors, BMP-2, BMP-4, BMP-6, BMP-12, sonic hedgehog, GDF5, GDF6, GDF8,PDGF, recombinant human growth and differentiation factor 5, tenascin-C,fibronectin, thromboelastin, thrombin-derived peptides, heparin-bindingdomains, demineralized bone matrix, platelet rich plasma, bone marrowaspirate, and bone fragments.
 13. The method of claim 1, where saidreinforced absorbable multilayered fabric further comprises cellsselected from the group consisting of bone marrow cells, stromal cells,stem cells, embryonic stem cells, chondrocytes, osteoblasts, osteocytes,osteoclasts, fibroblasts, pluripotent cells, chondrocyte progenitors,endothelial cells, macrophages, leukocytes, 5 adipocytes, monocytes,plasma cells, mast cells, umbilical cord cells, postpartum derivedcells, mesenchymal stem cells, epithelial cells, myoblasts, andprecursor cells derived from adipose tissue.